LOCUS       sequence-398269-        6350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of HsFbxo7-129-398 and HsSkp1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6350)
  AUTHORS   Kraus F, Goodall EA, Smith IR, Jiang Y, Paoli JC, Adolf F, Zhang J, 
            Paulo JA, Schulman BA, Harper JW
  TITLE     PARK15/FBXO7 is dispensable for PINK1/Parkin mitophagy in iNeurons 
            and HeLa cell systems.
  JOURNAL   EMBO Rep. 2023 Aug 3;24(8):e56399. doi: 10.15252/embr.202256399. 
            Epub 2023 Jun 19.
  PUBMED    37334901
REFERENCE   2  (bases 1 to 6350)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "EMBO Rep.";
            date: "2023-08-3"; volume: "24(8):e56399. doi"; pages: " 
            10.15252/embr.202256399. Epub 2023 Jun 19"
COMMENT     Sequence Label: pGEX4T1-TEV-HsFbxo7-129-398/Skp1
FEATURES             Location/Qualifiers
     source          1..6350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..938
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     RBS             1819..1841
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2400..2422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2567..2585)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2653..2757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2758..2826,2827..3618)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2976..2995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3789..4377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4278..4297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4531..4548
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4621..4698
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4699..5781
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4718..4737)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5830..5835,5836..5853,5854..5860)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5868..5884
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5892..5908
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5892..5908
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5904..6350,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5924..5941)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5924..5940)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5933..5955)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatgaa aacctgtatt ttcagggagg cgctagcgcc cagtctggtg
      961 tttggaatga cgacagtatg ttagggccta gtcaaaattt tgaagctgag tcaattcaag
     1021 ataatgcgca tatggcagag ggcacaggtt tctatccctc agaacccatg ctctgtagtg
     1081 aatcggtgga agggcaagtg ccacattcat tagagacctt gtatcaatca gctgactgtt
     1141 ctgatgccaa tgatgccttg atagtgttga tacatcttct catgttggag tcaggttaca
     1201 tacctcaggg caccgaagcc aaagcactgt ccatgccgga gaagtggaag ttgagcgggg
     1261 tgtataagct gcagtacatg catcctctct gcgagggcag ctccgctact ctcacctgtg
     1321 tgcctttggg aaacctgatt gttgtaaatg ctacactaaa aatcaacaat gagattagaa
     1381 gtgtgaaaag attgcagctg ctaccagaat cttttatttg caaagagaaa ctaggggaaa
     1441 atgtagccaa catatacaaa gatcttcaga aactctctcg cctctttaaa gaccagctgg
     1501 tgtatcctct tttggctttt acccgacaag cactgaacct accagatgta tttgggttgg
     1561 tcgtcctccc attggaactg aaactacgga tcttccgact tctggatgtt cgttccgtct
     1621 tgtctttgtc tgcggtttgt cgtgacctct ttactgcttc aaatgaccca ctcctgtgga
     1681 ggtttttata tctgcgtgat tttcgagaca atactgtcag agttcaagac acagattgga
     1741 aagaactgta caggaagtga taagtcgact cgagcggccg cctcgacgag accacaacgg
     1801 tttccctcta gaaataattt tgtttaactt taagaaggag atataccatg ccgtcaatta
     1861 agttgcagag ttctgatgga gagatatttg aagttgatgt ggaaattgcc aaacaatctg
     1921 tgactattaa gaccatgttg gaagatttgg gaatggatga tgaaggagat gatgacccag
     1981 ttcctctacc aaatgtgaat gcagcaatat taaaaaaggt cattcagtgg tgcacccacc
     2041 acaaggatga ccctcctcct cctgaagatg atgagaacaa agaaaagcga acagatgata
     2101 tccctgtttg ggaccaagaa ttcctgaaag ttgaccaagg aacacttttt gaactcattc
     2161 tggctgcaaa ctacttagac atcaaaggtt tgcttgatgt tacatgcaag actgttgcca
     2221 atatgatcaa ggggaaaact cctgaggaga ttcgcaagac cttcaatatc aaaaatgact
     2281 ttactgaaga ggaggaagcc caggtacgca aagagaacca gtggtgtgaa gagaagtgag
     2341 cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     2401 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2461 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     2521 cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc
     2581 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2641 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2701 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2761 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2821 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2881 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2941 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3001 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3061 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3121 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3181 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3241 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3301 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3361 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3421 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3481 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3541 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3601 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3661 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3721 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3781 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3841 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3901 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3961 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4021 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4081 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4141 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4201 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4261 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4321 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4381 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4441 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4501 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc
     4621 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     4681 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     4741 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     4801 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     4861 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     4921 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     4981 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     5041 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     5101 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     5161 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     5221 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     5281 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     5341 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     5401 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     5461 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     5521 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     5581 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     5641 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     5701 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5761 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     5821 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     5881 acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac
     5941 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     6001 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     6061 gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa
     6121 gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga
     6181 tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc
     6241 cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa
     6301 gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
